Tumor necrosis factor (TNF-α) is a potent cytokine having pro-inflammatory properties that is released by many cell types when stimulated. Studies have shown a relationship between elevated levels of TNF-α and a variety of diseases including septic shock, hematopoiesis, tumors, and inflammatory disorders of the central nervous system including HIV, encephalitis, cerebral malaria, and meningitis. Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Creutzfeldt-Jacob disease also are reportedly associated with enhanced TNF-α levels. See, e.g., Arvin et al., “The Role of Inflammation and Cytokines in Brain Injury,” Neuroscience and Biobehavioral Reviews, Vol. 20, No. 3 (1996), at pp. 445-452. Accordingly, various classes of compounds have been researched and developed to inhibit TNF-α production at both transcriptional and translational levels, e.g., corticosteroids, rolipram (a phosphodiesterase IV inhibitor suppressing TNF-α mRNA synthesis), calphostin, and imidazole-type cytokine suppressing anti-inflammatory drugs (CSAIDs). See, e.g., Dinarello, “Role of Pro- and Anti-Inflammatory Cytokines During Inflammation: Experimental and Clinical Findings, Review, Vol. 0393-974X (1997), at pp. 91-103.
Recently, attention has focused on the role of nuclear factor-κB (NF-κB) in the activation pathway that leads to production of TNF-α and other inflammatory cytokines and gene types. Besides TNF-α, NF-κB modulates many genes involved in immune function and inflammation, including interleukin (IL)-2, IL-6, IL-8, IL-2Rα, GM-CSF, intercellular adhesion molecule (ICAM-1), and vascular cellular adhesion molecule-1 (VCAM-1). Thus, inhibition of NF-κB and/or its activation pathway provides a means for treating a wide range of diseases including autoimmune diseases, Alzheimer's disease, atherosclerosis, oncogenesis, and so forth. See, e.g., Baldwin, “The NF-κB and IκB Proteins: New Discoveries and Insights,” Annual Rev. Immunol. Vol. 14 (1996), at pp. 649-81; see also Christman et al., “Impact of Basic Research on Tomorrow's Medicine, The Role of Nuclear Factor-κB in Pulmonary Diseases,” Chest, Vol. 117 (2000), at pp. 1482-87.
NF-κB is a transcriptional activator, which plays a central role in regulating the transcription of a number of genes including those, which encode proteins, involved in inflammatory and immune responses. Representative examples of genes controlled by NF-κB include the cytokines tumor necrosis factor (TNF-α), IL-1β, IL-6, and IL-8; the adhesion molecules E-selectin and vascular cell adhesion molecule (VCAM)-1; and the enzyme nitric oxide (NO)-synthase (for reviews, see Siebenlist et al. Annu. Rev. Cell Biol. 10:405-455, 1994; Bauerle and Baltimore, Cell 87:13-20, 1997). Also, NF-κB has been shown to be inducible by several stimuli, in addition to mediators of immune function, such as UV irradiation, growth factors, and viral infection.
NF-κB transcription factor normally resides in the cytoplasm in unstimulated cells as an inactive complex with a member of the inhibitor κB (IκB) inhibitory protein family. The IκB class of proteins includes IκB-α, IκB-β, and IκB-ε all of which contain ankyrin repeats for complexing with NF-κB (for review, see Whiteside et al., EMBO J. 16:1413-1426, 1997). In the case of IκB-α, the most carefully studied member of this class, stimulation of cells with agents which activate NF-κB-dependent gene transcription results in the phosphorylation of IκB-α at serine-32 and serine-36 (Brown et al. Science, 267:1485-1488, 1995).
Potential inhibitors of NF-κB and/or the NF-κB pathway have been identified as including Interleukin-10, glucocorticoids, salicylates, nitric oxide, and other immunosuppressants. IκB is a cytoplasmic protein that controls NF-κB activity by retaining NF-κB in the cytoplasm. IκB is phosphorylated by the IκB kinase (IKK), which has two isoforms, IKK-1 (or IκB kinase α, IKKα) and IKK-2 (or IκB kinase β, IKKβ). Upon phosphorylation of IκB by IKK, NF-κB is rapidly released into the cell and translocates to the nucleus where it binds to the promoters of many genes and up-regulates the transcription of pro-inflammatory genes. Inhibitors of IKK can block the phosphorylation of IκB and further downstream effects, specifically those associated with NF-κB transcription factors. Glucocorticoids reportedly inhibit NF-κB activity by two mechanisms, i.e., upregulating IκB protein levels and inhibiting NF-κB subunits. Nitric oxide also reportedly inhibits NF-κB through upregulation of IκB. However, these mechanisms of interaction are complex; for example, production of nitric oxide in lymphocytes reportedly enhances NF-κB activity.
Phosphorylation of IκB-α is critical for its subsequent ubiquitination and proteolysis, upon which NF-κB is released from complexing with IκB. NF-κB can then translocate into the nucleus and ultimately, activate gene transcription (Finco et al., Proc. Natl. Acad. Sci. USA 91:11884-11888, 1994; Baldi et al. J. Biol. Chem. 271:376-379, 1996; Roff et al. J. Biol. Chem. 271:7844-7850, 1996). Substituting both serine-32 and serine-36 of IκB with alanine prevents signal-induced NF-κB activation and also results in an IκB (e.g. IκB-α), which is not phosphorylated, ubiquitinated, or proteolytically digested (Roff et al. J. Biol. Chem. 271:7844-7850, 1996). Analogous serines have been identified in both IκB-β and IκB-ε, and phosphorylation at these residues appears to regulate the proteolytic degradation of these proteins by a mechanism similar to that of IκB-α (Weil et al. J. Biol. Chem. 272:9942-9949, 1997; Whiteside et al. EMBO J. 16:1413-1426, 1997).
More particularly, upon phosphorylation, ubiquitination, and degradation of IκB, NF-κB is released from the IκB/NF-κB complex and allowed to translocate from the cytoplasm to the nucleus and activate a number of genes, particularly those involved in inflammatory and immune responses. Since NF-κB is of significant importance in inflammation and immune responses, inhibition of the signal-inducible phosphorylation of IκB can be an important target for novel anti-inflammatory and immune-related agents in the treatment of inflammatory and immune system-related diseases and disorders, as described herein.
IκB kinase (IKK) is a high molecular weight (500-900 kD) multisubunit enzyme which phosphorylates IκB-α at positions serine-32 and serine-36 and has been isolated from HeLa cells (Chen et al. Cell 84:853-862, 1996; Lee et al. Cell 88:213-222, 1997; DiDonato et al. Nature 388:548-554, 1997). Two catalytic subunits termed IKK-1 and IKK-2 of IKK have been identified, cloned, and demonstrated to be widely expressed in human tissues (DiDonato et al. Nature 388:548-554, 1997; Zandi et al. Cell 91:243-252, 1997; Mercurio et al. Science 278:860-866, 1997; Woronicz et al. Science 278:866-869, 1997; Li et al. J. Biol. Chem. 273:30736-30741, 1998; Regnier et al. Cell 90:373-383, 1997).
The IKK-1 and IKK-2 catalytic subunits of IKK are highly homologous, having 50% sequence identity and more than 70% sequence similarity. IKK-1 and IKK-2 are 85- and 87-kDa proteins, respectively. Both kinases contain a catalytic domain followed by a leucine zipper domain and a helix-loop-helix (HLH) domain (Mercurio et al. Science, 278:860-866, 1997). When one subunit is recombinantly expressed without the other subunit, either one is still able to catalyze the phosphorylation of IκB (Li et al. J. Biol. Chem., 273:30736-30741, 1998). Thus, IKK, either IKK-1 or IKK-2, can play an important role in signaling IκB for ubiquination and further degradation.
In addition, in vitro studies have demonstrated that the full length IKKβ can autophosphorylate and phosphorylate its substrate, IκBα, as well; however, neither the N-terminal kinase domain-containing form, nor the C-terminal HLH domain-containing form of IKK was capable of autophosphorylation (U.S. Pat. No. 6,077,701 to Chu et al.). U.S. Pat. No. 6,077,701 discloses that compounds which increase IKKβ activity and/or binding to IκB can be potential modulators of inflammatory disease.
Evidence that IKK is involved in the signal inducible degradation of IκB-α was provided by both anti-sense inhibition of IKK-1 and the use of dominant-negative, catalytically-inactive mutants of IKK-1 and IKK-2 (DiDonato et al. Nature 388:548-554, 1997; Mercurio et al. Science 278:860-866, 1997; Woronicz et al. Science 278:866-869, 1997). Both approaches abrogated cytokine- and lipopolysaccharide (LPS)-induced activation of NF-κB. These in vitro assays implicate a role for IKK in activating NF-κB.
There are other kinases which can phosphorylate IκB and which have been implicated in the activation of NF-κB. For example, two kinases (pp90rsk and IKK-ε) have been demonstrated to phosphorylate IκB-α at serine-32 and/or serine-36. The overexpression of these kinases, and the use of dominant negative mutants to these kinases, have indicated a role for them in the phosphorylation of IκB in cells (Ghoda et al., J. Biol. Chem. 272:21281-21288, 1997; Peters et al., Mol. Cell. 5:513-522, 2000). The existence of multiple IκB kinases is indicative of redundant signaling pathways. Therefore, it is possible that an inhibitor of IKK-1 and/or IKK-2 can not necessarily show anti-inflammatory or immunosuppressive effects due to redundant signaling pathways in at least some cells.
Several in vitro types of studies have been performed to further investigate IKK and its properties. The in vitro types of cell biology studies (for example, overexpression of either IKK-1 or IKK-2, or of dominant negative versions of these kinases (DiDonato et al. Nature 388:548-554, 1997; Mercurio et al. Science 278:860-866, 1997; Woronicz et al. Science 278:866-869, 1997; Regnier et al. Cell 90:373-383; Zandi et al. Cell 91:243-252)) that appear to implicate IKK-1 and IKK-2 in NF-κB activation are sometimes artifactual. A particular example of such an artifactual study involves a kinase known as NF-κB-inducing kinase, NIK.
Overexpression of NIK in cells activated IKK and NF-κB, while expression of kinase-inactive forms of the enzyme blocked the stimulated activation of IKK and NF-κB (Malinin et al., Nature 385:540-544, 1997; Song et al., Proc. Nat. Acad. Sci. USA 94:9792-9796, 1997). Based on the foregoing, as well as on yeast two-hybrid studies showing a strong interaction between NIK and IKK (Regnier et al., Cell 90:373-383, 1997), NIK is suggested to be essential for the activation of IKK and, subsequently, NF-κB.
Additional studies have demonstrated that the NIK-IKK protein-protein interaction is important for NF-κB-dependent responses (WO99/43704; Publication Date: Sept. 2, 1999; Goeddel et al., U.S. Pat. Nos.: 5,851,812; 5,916,760; and 5,939,302). Goeddel et al. show that transient IKKβ overexpression induces luciferase reporter gene activity in both HeLa and 293 cells and overexpression of kinase-inactive IKKβ, which still associates with NIK, blocks activation. However, it was further determined that cells derived from mice deficient in NIK had no abrogated NF-κB activation (Karin and Ben-Nariah, Ann. Rev. Immunol., 18:621-663, 2000). Therefore, although some studies suggest the importance of NIK as a potential target for inhibiting NF-κB activation, the experimental results were not demonstrable in vivo, as reported by Karin and Ben-Nariah (supra). Thus, it is very important that in vitro-based and strictly cell biology-based experiments using overexpressed proteins always be cautiously interpreted.
Although there are several known synthetic inhibitors of IKK, none of these have played a successful role in vivo in disease inhibition. For example, aspirin (acetylsalicyclic acid) and salicylate have been demonstrated to be inhibitors of IKK-1 and IKK-2, but only at high, non-physiological concentrations which are much higher than those required to block prostaglandin synthesis through the inhibition of cyclooxygenase (Yin et al., Nature 396:77-80, 1998). Therefore, these agents are inappropriate for use in treatments or for testing the role of IKK in disease.
Similar to aspirin, 5-aminosalicyclic acid has been shown to inhibit IKK (Yan and Polk, J. Biol. Chem. 274:36631-36636, 1999), but this compound has several other activities, including the inhibition of prostaglandin and leukotriene synthesis (Peskar et al., Deg. Disc. Sci., 32:51S-56S, 1987; Horn et al., Scand. J. Gastroenterol. 26:867-879, 1991) which thereby precludes its use in testing the role of IKK in disease.
Sulindac is a cyclooxygenase inhibitor that has been demonstrated to also inhibit IKK-2 (Yamamoto et al., J. Biol. Chem. 274:27307-27314, 1999). However, in studies similar to those using aspirin, the concentrations of sulindac necessary for inhibiting IKK were much greater than those needed to inhibit cyclooxygenase. Therefore, this agent is also inappropriate for testing the role of IKK in disease.
Other inhibitors of IKK, more specifically of IKK-2, are cyclopentenone prostaglandins, such as 15dPGJ2 (Rossi et al., Nature 403:103-108, 2000). However, 15dPGJ2, can also activate peroxisome proliferator-activated receptor-gamma (PPAR-γ), a nuclear receptor that interferes with NF-κB transcriptional activity. Therefore, any anti-inflammatory effect could be explained by PPAR-γ activity rather than IKK inhibition. Thus, this inhibitor is not specific for inhibiting the downstream effects of NF-κB-induced inflammation via IKK.
Arsenite, another IKK inhibitor, is a reactive environmental toxin that has been shown to inhibit both IKK-1 and IKK-2 (Kapahi et al., J. Biol. Chem., 275:36062-36066, 2000). Due to its toxic effects, the therapeutic benefits or in vivo treatment utility of arsenite would therefore preclude its use in patients.
U.S. Pat. Nos. 4,229,452 and 4,200,750 to Warner et al., Colotta et al. (Eur. J. Med. Chem. 30:133-139, 1995) and Ceccarelli et al. (Eur. J. Med. Chem. 33:943-955, 1998; WO97/17079; May 29, 1997) disclose compounds that are structurally different from compound 6 as described herein.
The Aventis Pharma Deutschland GMBH(WO 01/68648) publication discloses particular substituted beta-carboline compounds. The WO 01/68648 publication does not disclose the IKK inhibitor compound(s) of the present invention, which have been shown to function and have proven to be effective in a variety of in vivo animal models of diseases or pathologies associated with inflammation, e.g. animal models of arthritis, inflammatory bowel disease, and pulmonary inflammation, and graft survival.
The Astrazeneca AB (WO 01/58890) publication discloses particular heteroaromatic carboxamide derivatives that are structurally different from the compounds of the present invention. The compounds of the WO 01/58890 publication do not demonstrate in vivo efficacy of the IKK inhibitor compounds of the present invention in animal models of disease associated with inflammation and/or the immune system.
In animal models of disease (i.e. knock-out mice) designed to test inhibition of IKK, the deletion of IKK-1 or IKK-2 has been demonstrated to be embryonic lethal (Li et al. Science 284:321-325, 1999; Hu et al. Science 284:316-320, 1999). Therefore, the use of IKK knockout mice to demonstrate a role of IKK in disease is neither practical nor feasible. In contrast, the present invention demonstrates for the first time that inhibitors of the catalytic activity of IKK-1 and IKK-2 are effective in murine models of disease. Such models are believed to be predictive of similar effects in human patients. Therefore, the treatment methods, in vivo models, and effectiveness of IKK inhibitors as described herein, are extremely beneficial and advantageous for the advancement of discovering and employing therapeutics for inflammation and immune system diseases. These animal models are therefore important tools for studying human diseases, specifically inflammatory and immune-related diseases.
Lactam-based tetracyclic compounds useful as antagonists of NMDA(N-methyl-D-aspartate) and AMPA (α-3-hydroxy-5-methylisoxazole-4-propionate) receptors are disclosed in WO 94/07893, Preparation of 5H, 10H-imidazo[1,2-a]indeno[1,2-e]pyrazine-4-one AMPA/KA Receptor Antagonist, filed by Aloup et al; and in articles by Mignani, Aloup, et al., “Synthesis and Pharmacological Properties of 5H, 10H-imidazo[1,2-a]indeno[1,2-e]pyrazine-4-one, a New Competitive AMPA/KA Receptor Antagonist,” Drug. Dev. Res., Vol. 48 (3) (1999), at pp. 121-29, and “An Efficient Preparative Route to Fused Imidazo[1,2-a]-Pyrazin-4-one Derivatives,” Heterocycles, Vol. 50, No.1 (1999), at pp. 259-267. Compounds such as lactams that are claimed to be useful for blocking excitatory amino acid receptors found in the brain and spinal cord are shown in U.S. Pat. Nos. 5,153,196 and 5,196,421, both assigned to Eli Lilly and Company. Tricyclic compounds having amino-substituents claimed to be useful as brain receptor ligands are disclosed in U.S. Pat. No. 5,182,386, assigned to Neurogen Corp., and in U.S. Pat. Nos. 4,160,097; 4,172,947; 4,191,766; 4,191,767; 4,198,508; 4,200,750; 4,225,724; and 4,236,015 in WO97/19,079, and in S. Ceccarelli et al, “Imidazo[1,2-a]quinoxalin-4-amines: A Novel Class of Nonxanthine A1-Adenosine Receptor Antagonists,” European Journal of Medicinal Chemistry Vol. 33, (1998), at pp. 943-955. To applicants' knowledge, 4-amino substituted tetracyclic compounds according to formula (I) have not been previously described.
As can be appreciated, those in the field of pharmaceutical research continue to seek to develop new compounds and compositions having increased effectiveness, bioavailability, and solubility, having fewer side effects, and/or providing the consumer with a choice of options. Particularly in the area of immune response, many individuals respond differently depending upon the type of treatment and chemical agent used. Mechanisms of action continue to be studied to aid in understanding the immune response and in developing compounds effective for treating immune-related disorders.
In view of the dearth of safe, effective, and novel therapeutics for treating inflammatory or immune-related diseases, there is a need for new anti-inflammatories and agents that reduce, eliminate, and/or ameliorate inflammation and immune-related disease. In addition, the discovery of additional proteins involved in the processes of inflammation and immune-related diseases and disorders is important for controlling inflammation and adverse immune responses, processes, and the diseases and disorders related thereto. Thus, the identification and characterization of the proteins involved in IKK-mediated cellular processes would benefit the art and be advantageous for providing therapies for those suffering from such disorders and diseases.